1. Field of the Invention
This invention relates to a chemiluminescent assay for the determination of density or activity of intact viable (living) cells per se or of such cells in living tissue or organ for biological studies, which is convenient and exact.
2. Description of the Prior Art
The measurement of the growth rate of microorganisms, mammalian cells, and plant tissues is an important procedure in many biological assays. The viable cells can be measured by several staining methods which require several steps depending on the type of cells, but the staining methods are not useful for determining the activity of viable cells.
Recently, a colorimetric assay for cellular growth and survival using tetrazolium salt has been reported and is useful for measuring the activity of viable cells, in which MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromine] used for this assay is incorporated in viable cells, and a colored product is produced by active mitochondria (Mosmann, T. (1983) J. Immunol. Methods Vol. 65, pp. 55-63.
However, this procedure requires long incubation times. For example, mammalian cells are incubated with MTT for 4 hours at 37.degree. C., and the formazan produced must be solubilized by the addition of an acid solution to minimize the interference of phenol red contained in normal tissue culture medium.
On the other hand, a bioluminescent assay is used to determine the intracellular concentration of NAD(P)H or ATP(Hasting, J. W. (1978) in Methods in Enzymology (Colowick, S, P., and Kaplan, N. O., Eds.), Vol. 57, pp. 125-134, Academic Press, Orlando, Fla.; De Luca, M., and McElroy, W. D. (1978) in Methods in Enzymology (Colowick, S, P., and Kaplan, N. O., Eds.), Vol. 57, p. 3, Academic Press, Orlando, Fla.). The procedure requires the extraction of NAD(P)H of ATP from the cells, and all instruments must be carefully washed to avoid the contamination of NAD(P)H or ATP. The luciferase used for this assay is expensive and furthermore is unstable.